The authors have declared that no competing interests exist.
Conceived and designed the experiments: MS HC. Performed the experiments: MS. Analyzed the data: MS HC. Contributed reagents/materials/analysis tools: MS HC. Wrote the paper: MS HC. Software used in analysis: HC.
Aerobic anoxygenic phototrophs (AAPs) as being photoheterotrophs require organic substrates for growth and use light as a supplementary energy source under oxic conditions. We hypothesized that AAPs benefit from light particularly under carbon and electron donor limitation. The effect of light was determined in long-term starvation experiments with
Aerobic anoxygenic phototrophs (AAPs) are widespread in marine habitats
Comparable to purple bacteria, AAPs are capable of light-driven and respiratory electron transport for their energy metabolism. However, this activity is different in several aspects. AAPs use light energy under oxic conditions and contain much less bacteriochlorophyll
Light-driven proton translocation and ATP formation by AAPs have been investigated in several studies
The purpose of the present study was to identify the conditions under which AAPs benefit the most from their photosynthetic capacities. We hypothesized that AAPs benefit from photon energy under conditions of carbon and electron donor limitation, as proteomic responses to starvation and light conditions among AAPs have been reported
Batch cultures were incubated at 23°C on a shaker (Innova 42-R, New Brunswick, 125 rpm) in the dark (DD), under continuous illumination (LL; 12 µmol photons m−2 s−1) or light and dark cycles (LD, 12 h/12 h, 12 µmol photons m−2 s−1). For determining the optimum light intensity, GRO-LUX fluorescent light bulbs were used as light source. Growth was monitored by measuring the optical density (OD) at 650 nm. For starvation experiments, stationary phase cells were harvested by centrifugation at 3330×
Total cell counts were analyzed by SYBR Green staining and epifluorescence microscopy (Olympus BX51). For live counts, serially diluted samples were plated onto MB agar plates incubated at 25°C and counted after 4–8 weeks. To study morphological changes under different conditions, suspensions of starved cells were placed on a Formvar copper grid (
To determine the dry biomass, starved cells were harvested and washed with 50 mM ammonium acetate buffer and dried overnight at 80°C. Lowry’s method with Folins reagent was used to determine the total protein concentration
Washed cells were resuspended in HEPES buffer (10 mM, pH 7.75, supplemented with NaCl 20 g per liter, KCl 0.5 g per liter). Oxygen concentrations were measured with a Clark-type oxygen electrode (Bachofer, Reutlingen, Germany) while the chamber was maintained at 30°C. The influence of light on the respiration was checked by illuminating the cells in the reaction chamber using a halogen lamp at 400 µmol photons m−2 s−1.
Washed cell suspensions (3 ml) were incubated in Hungate tubes sealed with rubber stoppers and flushed with nitrogen at room temperature. The physiological responses were analysed by incubation of cell suspensions under anoxic conditions and studying the reaction upon oxygen and organic substrate addition in the dark and in the light. The energy content of the cells was determined using ATP bioluminescence Assay Kit CLS II (Boehringer Mannheim) and the extraction of ATP was carried out as described in
Washed cells were resuspended in 2.5 ml salt solution containing 2% NaCl and 0.05% KCl and treated with 500 µl of 0.5 M KSCN, thus providing the membrane-permeable anion SCN– in order to destroy the membrane potential. The cell suspensions were then flushed with nitrogen for 20 min. Known amounts of oxygen (16 nmol O2) in KCl solution, were injected into the measuring chamber equipped a with pH electrode (Mettler Toledo pH Electrode, Inlab Micro).
Cells pre-grown with succinate at different light intensities were harvested, washed, resuspended in medium without carbon source, and incubated under light and dark cycles (LD, 12 h/12 h) for four weeks. Different light intensities were tested to determine the optimum illumination for survival. Under all conditions, total and viable cell counts decreased However, cultures incubated at medium light intensity (12 µmol photons m−2 s−1) showed higher survival rates (
Light intensities (µmol m−2 s−1) | ||||
Days starved | Parameters | High | Middle | Low |
23 | 12 | 3 | ||
0 | Protein/biomass | 0.41±0.02 | 0.46±0.02 | 0.49±0.01 |
Total cell counts/ml (108) | 2.62±3 | 2.6±2 | 2.66±1.3 | |
Viable counts/ml (108) | 1.97±1.2 | 2.28±1.4 | 2.10±0.18 | |
BChl |
2.12±2 | 2.82±3 | 2.7±2 | |
Carotenoids (nmol/mg protein) | 1.39±0.03 | 1.88±0.07 | 1.87±0.11 | |
14 | Protein/biomass | 0.36±0.05 | 0.38±0.02 | 0.36±0.02 |
Total cell counts/ml (108) | 2.17±0.85 | 2.36±1.95 | 1.46±2 | |
Viable counts/ml (107) | 1.91±0.11 | 16.3±0.23 | 2.67±3.1 | |
BChl |
1.11±3 | 1.96±1 | 1.66±5 | |
Carotenoids (nmol/mg protein) | 1.17±0.03 | 1.26±0.52 | 1.11±0.05 | |
21 | Protein/biomass | 0.33±0.03 | 0.33±0.01 | 0.24±0.1 |
Total cell counts/ml (108) | 1.06 |
2.14±0.08 | 1.31±2 | |
Viable counts/ml (106) | 10.4 |
117.21±1.83 | 6.27±0.02 | |
BChl |
0.68±3 | 1.17±1 | 1.06±5 | |
Carotenoids (nmol/mg protein) | 0.76±0.11 | 0.61±0.03 | 0.42±0.07 |
a Cell counts were performed from single bottle as the biological replicate cells started to clump.
Starvation experiments were performed at the optimum light intensity (12 µmol photons m−2 s−1) under different light regimes, i.e. light and dark cycle (LD), continuous light (LL), and continuous dark (DD). LD conditions resulted in the highest survival rates. During the first week of starvation, total and viable cell counts of the DD cultures decreased faster than those in LL and LD cultures (
Symbols: total (solid lines) and viable (broken lines) counts of cells starved under light/dark cycles (LD, 12/12 h, squares), continuous light (diamonds), or in the dark (triangles). The light intensity was 12 µmol photons m−2 s−1. Mean values and standard deviation shown are from two biological replicates.
(A) Freshly grown cells of
The physiological fitness of cells was assessed by respiration rates, respiration-driven proton translocation, and the ability of ATP regeneration. Respiration rates served as a measure of physiological activity and light utilization. The slowdown of respiration in the light indicates saving of electron donors and light-driven energy conservation. Independent of the starvation conditions, endogenous respiration rates were 10% higher in the dark than under illumination during the early starvation phase. Addition of succinate as electron donor resulted in a similar effect. After 4 weeks of starvation only LD cells showed this effect (
Starvation conditions | Days starved | Endogenous respiration (nmol O2 min−1 (mg DW) −1) | ||
Dark | Light | Dark again | ||
3 | 23.78±1.2 | 20.79±2.3 | 21.89±0.9 | |
7 | 15.66±0.91 | 14.09±1.3 | 15.23±2 | |
21 |
2.09 | 2.09 | 2.09 | |
3 | 22.13±4 | 18.77±1.2 | 20.63±0.6 | |
7 | 17.57±2.1 | 16.35±0.83 | 17.01±1.9 | |
21 | 9.31±0.38 | 9.24±2 | 9.21±2 | |
3 | 27.63±4.3 | 25.01±2.8 | 25.83±0. 5 | |
7 | 19.87±2.5 | 17.21±0.68 | 18.19±0.41 | |
21 |
4.75 | 4.75 | 4.75 | |
3 | 22.68±2.8 | 22.06±3.1 | 21.33±2.6 | |
7 | 18.59±1.8 | 17.96±1 | 18.12±1 | |
21 | 9.99±0.95 | 9.19±1.1 | 9.39±0.2 |
[Rates were measured in the dark, after switching on light (400 µmol photons m−2 s−1) for 2 min, and in the dark again].
a Respiration rates were performed from single bottle as the other harvested cells started to clump after anoxygenic incubation.
Utilization of light was also shown in proton translocation measurements, when oxygen and light were supplied simultaneously to cells incubated under N2 (
Cells were maintained in the dark (dark bar) and the influence of light on the number of translocated per added oxygen atom (H+/O) were studied when cells were illuminated (grey bar). (A) Cells starved under continuous light (LL); (B) cells starved under light and dark cyclers (LD). Light intensity: 400 µmol photons m−2 s−1. Mean values and standard deviation shown are from two biological replicates.
The cytoplasmic ATP concentrations showed a steady decrease upon starvation (
(10−18) mol ATP per cell | (10−18) mol ATP per cell | |||||
Days starved | LL | LL+N2 | O2+ light | LD | LD+N2 | O2+ light |
3 | 3.02±0.2 | 0.01±0.2 | 2.66±0.2 | 2.95±0.2 | 0.58±0.009 | 2.25±0.2 |
7 | 1.9±0.1 | 0.03±0.0 | 1.47±0.06 | 2.21±0.2 | 0.005±0.02 | 1.71±0.02 |
14 | 0.91±0.1 | 0.05±0.1 | 0.61±0.02 | 1.09±0.01 | 0.0006±0.01 | 0.9±0.04 |
21 | 0.22±0.2 | 0.01±0.0 | 0.09±0.1 | 0.45±0.04 | 0.004±0.001 | 0.21±0.01 |
(ATP was measured after harvesting the cells, after anoxic incubation for 2 h, and after flushing the suspension with air in the light [400 µmol photons m−2 s−1, 2 min].
Under DD conditions, total cell counts in complex media were stable over six weeks (
Our study demonstrated that day and night cycle substantially increases the survival of
All measured parameters show that
Not only continuous light, but also high intensities during day and night cycles had negative effects on the cells. The harmful light effects were also recognized from the pigment analysis, as cells increased their carotenoid contents at high light intensities likely as a protective response. By low pigment concentrations, AAPs might prevent formation of reactive oxygen species
The light-supported survival of starvation of
Considering the specific advantage of the AAPs in their natural environment, it becomes clear that light is not the overall limiting factor for their distribution. Cottrell et al. found that AAPs are distributed over the whole photic zone
(PPT)
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We thank Feng Chen (Institute of Marine and Environmental Technology, University of Maryland), Irene Wagner-Döbler (HZI, Braunschweig) and Marwan Majzoub (University of New South Wales). We also extend our gratitude to Bert Engelen, Verona Vandieken and Matthias Wietz from the ICBM for giving valuable comments. We thank Michael Pilzen and Sonja Standfest for technical assistance.